Evaluation of Minichromosome Maintenance-3 (MCM3) in Oral Squamous Cell Carcinoma.

STATEMENT OF THE PROBLEM
The expression of minichromosome maintenance-3 (MCM3) proteins and their diagnostic value in oral mucosal dysplasia and squamous cell carcinoma (SCC) is not well known.


PURPOSE
This study was conducted to evaluate the usefulness of minichromosome maintenance 3 (MCM3) as a biomarker for diagnosis of oral premalignant lesions and SCC.


MATERIALS AND METHOD
In this study, 31 oral SCC, 20 dysplastic epithelium and 20 controls were selected and immunohistochemical staining was done for MCM3. ANOVA, Tukey HSD, Mann-Whitney and Kruskal-Wallis tests were used to compare the groups and the correlation between different grades.


RESULTS
There was increasing trend of MCM3 from control to dysplastic epithelium and from dysplastic epithelium to SCC both in suprabasal layers and in total epithelial layers. MCM3 expression was elevated with increasing the grade of dysplasia, but there was no statistically significant difference (p= 0.93). The expression was also increased in high grades of SCC compared to lower grades.


CONCLUSION
MCM3 can be used as a useful biomarker in the diagnosis of premalignant lesions and oral SCC.


Introduction
Proliferation markers have been broadly used to detect various human malignancies. Conventional proliferation markers such as Ki-67 and proliferation cell nuclear antigen (PCNA) are the most largely used proliferation markers in diagnosis of several human malignancies such as breast tumors, sarcoma in soft tissues, meningioma, malignancies of prostate, and non-Hodgkin lymphoma. [1][2][3][4] Minichromosome maintenance (MCM) proteins are essential factors for replication of DNA which were initially identified in Saccharomyces cerevisiae. [5] MCM [2][3][4][5][6][7] proteins are best known among this family and are critical components of the replication initiation complex which initiates synthesis of DNA in eukaryotes. [6][7] Origin recognition complex (ORC) is a protein complex with the ability of binding to the origins of replication and forming a landing pad for the replication factors Cdc 6 and Cdt 1 . At this time, MCM s (MCM [2][3][4][5][6][7] are recruited to the chromatin. So, the pre-replication complex (pre-RC) is formed which allows S-phase to be initiated. After S-phase entry, this complex is disassembled. MCM proteins and Cdc 6 leave the chromatin following the increased activity of cyclin A-CDK 2 (cyclindependent kinase2). Cdc 6 is carried to the cytoplasm and Cdt 1 is proteolysed. Any Cdt 1 that has escaped proteolysis will bind to geminin. [8] Previous experiments have demonstrated that MCM proteins 2, 4, 5, 6 and 7 are associated with several cancers. [9][10][11][12] Only a few experiments have been done on MCM 3 compared to other members of this family. The purpose of this study was to evaluate the expression of MCM 3 proteins and their diagnostic value in oral mucosal dysplasia and squamous cell carcinoma (SCC).

Materials and Method
Tissue samples (n=70) were selected from the archive of oral pathology department of the Shiraz Dental School and Khalili Hospital. Twenty cases of dysplastic epithelium from lesions diagnosed as leukoplakia (including mild (n=8), moderate (n=8) and severe (n=4)) and SCC (n=31) were selected. Normal squamous epithelium was obtained from lesions diagnosed as irritation fibrosis (n=20). SCC patients had not received any chemotherapy or radiotherapy.
Hematoxylin and eosin stained slides were examined by three pathologists and cases with adequate tissues samples without hemorrhage and necrosis and similar degrees of inflammation were selected for immunohistochemistry (IHC) staining.

Immunohistochemical Analysis
All specimens were fixed in 10% formalin and routine

Statistical analysis
Statistical analysis was carried out using SPSS Software, version 18. Interclass correlation coefficient (ICC) was used to evaluate the intra-observer error (ICC=0.892). ANOVA and Tukey HSD analysis were used to compare the groups. Mann-Whitney and Kruskal-Wallis test were used to evaluate the correlation between different grades. Differences were considered to be statistically significant at p< 0.05.

Results
Clinical features of the patients are summarized in Table   1. Oral SCC specimens were composed of 31 patients In dysplastic epithelium, MCM 3 positively stained nuclei were present above the basal layer, whereas in SCC samples, nuclear staining was seen in superficial layers ( Figure 1).  Figure 2).
Score staining showed no significant difference between dysplastic epithelium and SCC (p= 0.11) and also between normal epithelium and SCC (p= 0.10).
In samples of dysplastic epithelium, MCM 3 expression increased gradually and became stronger from mild dysplasia to severe dysplasia ( Figure 3) but did not have significant difference (p= 0.93).
In SCC cases, the expression of MCM 3 protein gr-   showed in 2008 that a complete complex of MCM is necessary to protect the integrity of genome against natural replication stress during S phase. [21] So, the higher expression of MCM3 in higher grades of dys-plastic in our study may propose the same defensive mechanism against genomic injury and before malignant transformation. [18] In SCC, most of the differentiated tumor cells showed higher expression and score staining for MCM3 similar to the result of Gan et al.'s study in 2010, [13] which showed positive correlation between the tumor differentiation and MCM3 expression in cervical SCC. They also reported higher expression of MCM3 in poorly differentiated lesions.

Conclusion
In conclusion, findings of the present study showed that MCM3 could be used as a useful marker in diagnosis of premalignant lesions and SCC of oral cavity.